Immunotherapeutic modulation of amyloidogenic disease using non-fibrillogenic, non-amyloidogenic polymerized proteins and peptides

ABSTRACT

The present invention is directed to polymerized products and compositions useful for the treatment and prevention of amyloid disease in a subject. The invention further relates to isolated antibodies that recognize a common conformational epitope of amyloidogenic proteins or peptides that are useful for the diagnosis, treatment, and prevention of amyloid disease.

This application is a continuation of U.S. patent application Ser. No.13/550,316, filed Jul. 16, 2012, which claims the benefit of U.S.Provisional Patent Application Ser. Nos. 61/509,320 and 61/509,442, bothfiled Jul. 19, 2011, which are hereby incorporated by reference in theirentirety.

This invention was made with government support under grant numbersAG20245 and NS073501 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical agents and antibodiessuitable for the diagnosis, prevention, and treatment of amyloiddisease.

BACKGROUND OF THE INVENTION

Amyloidosis broadly encompasses a variety of diseases that arecharacterized by the extracellular or intracellular deposition ofamyloid proteins in tissues and/or organs. Amyloids are insolublefibrous protein/peptide aggregates and their deposition may occur inlocalized sites or systemically. The fibrillar composition of thesedeposits is an identifying characteristic for the various forms ofamyloid disease. In some cases the amyloid protein/peptide accumulatesintracellullary, resulting in cell dysfunction and ultimately celldeath. Examples of intracellular amyloid proteins include α-synuclein,forming Lewy bodies in Parkinson's disease, and huntingtin, formingneuronal inclusions in Huntington disease. The pathogenesis ofAlzheimer's disease (AD), the most common of the amyloid relatedneurodegenerative disorders, is linked to the cleavage of the amyloidprecursor protein generating the amyloid-β (Aβ) peptide which undergoesa shape change into a pathological conformer having a high β-sheetcontent. Intracerebral and cerebrovascular deposits composed primarilyof fibrils of the pathological Aβ peptide are characteristic of bothfamilial and sporadic forms of AD. In addition to Aβ, abnormally foldedand phosphorylated tau protein forms toxic oligomeric structures andneurofibrillary tangles in AD. Similar to AD, prion-associated diseases,such as Creutzfeld-Jacob disease, have also been characterized asamyloid diseases. The pathogenesis of prion disease is linked to achange of the cellular prion protein (PrP^(C)) into the diseaseassociated PrP^(Sc) (Sc for scrapie). Currently, there is no effectivetherapy for any of these disorders.

An active area of translational research and current clinical trials foramyloid disease has focused on immunotherapy, using both passive andactive immunization against amyloid proteins, particularly Aβ in AD(Wisniewski et al., “Amyloid-β Immunization for Alzheimer's Disease,”Lancet Neurol 7:805-811 (2008)). Although immunotherapy holds greatpromise as a means of reducing amyloid deposition, it, unfortunately,has been accompanied by major obstacles. Specific problems associatedwith immunotherapy that were identified in a clinical trial for ADinclude the potential of toxicity from encephalitis (related toexcessive cell mediated immunity), the immunological targeting of boththe normal and abnormal Aβ peptide, the failure to address tau relatedpathology, and the apparent poor efficacy. Moreover, although autopsydata from this early immunotherapy vaccine trial suggested that manypatients had a significant reduction in amyloid burden, these patientsexhibited only minor cognitive benefits (Wisniewski et al., “Amyloid-βImmunization for Alzheimer's Disease,” Lancet Neurol 7:805-811 (2008)and Holmes et al., “Long Term Effects of Aβ42 Immunization inAlzheimer's Disease: Immune Response, Plaque Removal and ClinicalFunction,” Lancet 372:216-223 (2008)). Therefore, an immunotherapeuticapproach that can effectively reduce amyloid burden and overcome theaforementioned problems is warranted.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a non-amyloidogenic,non-fibrillogenic polymerized product comprising two or more proteinand/or peptide units, where each unit is independently selected from thegroup consisting of an amyloid-beta (Aβ) peptide, an α-synuclein proteinor peptide, a tau protein or peptide, a TAR DNA-binding protein 43(TDP-43) protein or peptide, an amylin protein or peptide, a prionprotein (PrP) protein or peptide, and any combination thereof.

A second aspect of the present invention is directed to an isolatedantibody or binding portion thereof having antigenic specificity for anepitope a non-amyloidogenic, non-fibrillogenic polymerized product. Thepolymerized product comprises two or more protein and/or peptide units,each unit independently selected from the group consisting of anamyloid-beta (Aβ) peptide, an α-synuclein protein or peptide, a tauprotein or peptide, a TAR DNA-binding protein 43 (TDP-43) protein orpeptide, an amylin protein or peptide, a prion protein (PrP) protein orpeptide, and any combination thereof.

The development of an effective immunotherapeutic approach for theprevention and treatment of amyloid related diseases has been hinderedby potential cell-mediated toxicity, non-specific immunologicaltargeting of both normal and amyloidogenic proteins, and overall poorefficacy. The immunotherapeutic approach of the present inventionemploys non-amyloidogenic, non-fibrillogenic polymerized protein and/orpeptides and co-polymerized proteins and/or peptides to generate aspecific immunological response to conformational epitopes that areshared by various amyloidogenic proteins, thereby overcoming many of theabove noted obstacles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the results of locomotor activity testing in SwDItransgenic (Tg) control mice and Tg mice vaccinated with syntheticpolymerized Aβ peptide (Aβ1-30₁₈K₁₉K), polymerized ABri peptide(ABri-Glut), or polymerized Aβ1-42 peptide. No significant differencesbetween the groups were noted in distance traveled (FIG. 1A) or meanvelocity (Vmean) (FIG. 1C). Aβ1-42 vaccinated Tg mice exhibited slowermaximum velocity (Vmax) (FIG. 1B) (p=0.0234 post-hoc only; Aβ1-42 versuscontrol p<0.05) and longer resting time (FIG. 1D) (p=0.0311 post-hoconly; Aβ1-42 versus control p<0.05) compared to controls. No significantdifferences between the Aβ1-30₁₈K₁₉K and ABri treated Tg animals andcontrols in these parameters were observed (FIGS. 1B and 1D)

FIGS. 2A-2D show the results of locomotor activity testing in Tg3xtransgenic control mice and Tg mice vaccinated with syntheticpolymerized Aβ peptide (Aβ1-30K₁₈K₁₉), polymerized ABri peptide(ABri-Glut), or the combination of Aβ1-30K₁₈K₁₉ and ABri polymerizedpeptides. No significant differences between the groups were noted indistance traveled (FIG. 2A), maximum velocity (Vmax) (FIG. 2B), meanvelocity (Vmean) (FIG. 2C), or in resting time (FIG. 2D).

FIGS. 3A-3C illustrate the effects of polymerized peptide vaccination onbehavioral improvement in 3×Tg and SwDI transgenic animal models ofamyloidogenic disease. FIG. 3A is a graph depicting the results of theradial arm maze. The number of errors (y-axis) versus the day of testing(x-axis) are plotted for 3×Tg mice vaccinated with polymerized ABri,transgenic control (Tg control), and wildtype. Vaccinated transgenicanimals commit significantly fewer errors that non-treated transgenicand perform similarly to the wildtype animals. p=0.0044 one-way ANOVA;p<0.01 wildtype and Tg pABri treated vs. Tg control; p=>0.05 wildtypevs. Tg pABri treated. FIG. 3B is a similar graph showing the radial armmaze results for 3×Tg animals vaccinated with polymerized ABri,polymerized Aβ1-30K₁₈K₁₉, and the combination (polymerized ABri andAβ1-30K₁₈K₁₉). All treatment groups committed significantly fewer errorsthan the transgenic control animals. p=0.004 by one-way ANOVA; p<0.01all Tg Treated groups and wild-type versus Tg Control; no significantdifference between wild-type and Tg treated groups. FIG. 3C is anothergraph depicting the results of the radial arm maze cognitive testing. Inthis graph the number of errors (y-axis) versus the day of testing(x-axis) are plotted for SwDI transgenic mice vaccinated withpolymerized ABri, polymerized Aβ1-30₁₈K₁₉K, polymerized Aβ1-42,transgenic control mice, and wildtype mice (WT). All treatment groupscommitted significantly fewer errors than the transgenic control animals(p<0.01 all Tg Treated groups and wild-type versus Tg Control).

FIGS. 4A-4C are bar graphs showing IgM and IgG antibody levels raised inTgSwDI mice vaccinated with polymerized ABri peptide (pABri) (FIG. 4A),polymerized Aβ1-30K₁₈K₁₉ (FIG. 4B), and polymerized Aβ1-42 peptide (FIG.4C). Antibody titers against Aβ1-40, Aβ1-42, pABri, and a mutantAβ1-30KK were measured in vaccinated mice prior to the first inoculation(T0), after the 6th inoculation (T1) and at the time of sacrifice (TF).(***p<0.0001, **p<0.01, *p>0.05 versus T0).

FIGS. 5A-5D are bar graphs showing IgM and IgG antibody levels raised in3×Tg mice vaccinated with polymerized ABri peptide (FIG. 5A),polymerized Aβ1-30K₁₈K₁₉ (FIG. 5B), the combination of polymerizedpeptides (FIG. 5C), and vehicle (FIG. 5D). Antibody titers againstAβ1-40, Aβ1-42, pABri, and a mutant Aβ1-30KK were measured in vaccinatedmice prior to the first inoculation (T0), after the 6th inoculation(T1), and at the time of sacrifice (TF). (***p<0.0001, **p<0.01, *p>0.05versus T0).

FIGS. 6A-6C depict the amyloid and paired helical fiber (PHF1) burden inTg3x transgenic control and vaccinated animals. FIG. 6A is a bar graphshowing the reduction of hippocampal amyloid burden in Tg3x animalsadministered polymerized ABri (Tg-pABri), polymerized Aβ1-30₁₈K₁₉K(Tg-AB1-30KK), or the combination of polymerized peptides (Tg-combined)compared to transgenic control (Tg-control) animals (p=0.001 by one wayANOVA post-hoc testing; p<0.01 all treatment groups vs. control). FIGS.6B and 6C show a reduction in PHF1 in the hippocampus and cortex,respectively, of Tg3x animals administered polymerized ABri or thecombination of polymerized ABri and Aβ1-30₁₈K₁₉K (p<0.0001 one way ANOVApost hoc; p<0.001 Tg-ABri and Tg-combined vs. Tg-control andTg-Aβ1-30₁₈K₁₉K for both hippocampal and cortical analyses). Nosignificant difference was found between Tg-control and Tg-Aβ1-30₁₈K₁₉Ktreated animals.

FIGS. 7A-7B depict the amyloid burden in SwDI transgenic control andvaccinated animals. FIG. 7A is a bar graph showing the reduction inhippocampal amyloid burden in polymerized ABri vaccinated (Tg pABri),polymerized Aβ1-30₁₈K₁₉K vaccinated (Tg-Aβ1-30KK), and polymerizedAβ1-42 vaccinated (Tg-Aβ42) animals compared to transgenic control (TgControl). FIG. 7B is an electron micrograph (EM) image of negativelystained polymerized Aβ1-42 peptide, which is predominately in the formof spherical particles of ˜200 nm.

FIGS. 8A-8B are EM images of aged Aβ1-42 peptides. In FIG. 8A Aβ1-42peptide was aged for 3 months after controlled polymerized withglutaraldehyde. There is no evidence of fibril formation, and the samplewas typical of oligomerized peptides/proteins. FIG. 8B is an Aβ1-42peptide from the same synthesis batch as the peptide in FIG. 8A. Thispeptide was dissolved in saline and aged for 3 months before the EM. Incontrast to the polymerized Aβ1-42 peptide, the unpolymerized Aβ1-42sample was completely fibrillized.

FIG. 9 shows specific anti-PrP IgA antibodies in feces of control anddeer vaccinated with prion protein (PrP) as measured by ELISA. Whitetail deer were orally inoculated with an attenuated salmonella carryingdeer-PrP or an empty vector (control) four times over a period of a fewmonths following the protocol for mucosal vaccination described in Goniet al., “High Titers of Mucosal and Systemic Anti-PrP AntibodiesAbrogate Oral Prion Infection in Mucosal-Vaccinated Mice,” Neurosci.153:679-686 (2008), which is hereby incorporated by reference in itsentirety. Animals were subsequently orally boosted with a mixture ofpolymerized PrP and polymerized recombinant PrP peptides as described inthe Examples herein. Antibody titers were measure before the firstinoculation (T0), after the fourth inoculation (T5), and ten days afterthe boost (T6). The boost with PrP and polymerized PrP fragments wasrepeated two months after the first boost, and titers were againmeasured 10 days after the second boost (T7). The control group did notdevelop any noticeable titer whereas the vaccinated group showed someincrease in mucosa titer after the immune response was established withthe salmonella oral delivery. The titers were very low (T5) but weregreatly enhanced after the animals were boosted with the polymerized PrPand PrP fragments (T6 and T7) showing the importance of these antigenicpreparation to have a sustainable immune response.

FIG. 10 shows serum anti-PrP IgM antibodies of control deer and deervaccinated with PrP as described in FIG. 9 . Again the control group didnot show any noticeable increase in antibody titer; whereas thevaccinated group showed some concomitant IgM titer in serum at the sametime of the mucosal response (T5). Anti-PrP antibody titers in thevaccinated animals greatly increased after the two boosts with thepolymerized PrP and PrP fragments showing that this type of boost couldinvoke a therapeutic serum response. Vaccinated vs. control at T0 notsignificant, * p<0.05, ** p=0.0009, *** p<0.0001 (two-tailed test).

FIG. 11 shows immunoblots of salmonella lysate (lane 1), sheep PrP (lane2), polymerized sheep PrP (lane 3), deer PrP (lane 4), and polymerizeddeer PrP (lane 5) that were developed using purified antibodies fromvaccinated animal 781 at T7 and control animal 786 at T7. Both animalsmounted a good immune response to salmonella (lane 1); however, only thevaccinated animal had antibodies against different deer PrP molecules(lane 4) and the oligomers present in the polymerized deer PrP (lane 5).Vaccinated vs. control at T0 not significant, * p<0.017, ** p=0.0022,*** p<0.01 (two-tailed test).

FIG. 12 is a Kaplan and Meier survival curve showing the protectiveeffect of PrP vaccination in White Tail Deer at 18 months afterchallenge. Three out of six control animals became sick with theprionoses Chronic Wasting Disease and were properly euthanized. None ofthe vaccinated animals had signs of the disease at 18-months. Protectionfor the progression of the disease is evident and due to the immuneresponse elicited by the inoculations involving the polymerized PrP andPrP fragments.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a non-amyloidogenic,non-fibrillogenic polymerized product comprising two or more proteinand/or peptide units, where each unit is independently selected from thegroup consisting of an amyloid-beta (Aβ) peptide, an α-synuclein proteinor peptide, a tau peptide or peptide, a TAR DNA-binding protein 43(TDP-43) protein or peptide, an amylin protein or peptide, a PrP proteinor peptide, and any combination thereof.

In accordance with this aspect of the invention suitable Aβ peptides ofthe polymerized product include, without limitation, Aβ1-42 and peptidesderived therefrom. The amino acid sequence of Aβ1-42 is provided as SEQID NO:1 below.

(SEQ ID NO: 1) daefrhdsgy evhhqklvff aedvgsnkga iiglmvggvv ia

Accordingly, in one embodiment of the invention, the Aβ peptide of thepolymerized product comprises amino acid residues 1-42 of SEQ ID NO:1.Alternatively suitable Aβ peptides of the polymerized product include,without limitation, peptides comprising amino acid residues 1-16 of SEQID NO:1, amino acid residues 1-20 of SEQ ID NO: 1, amino acid residues1-30 of SEQ ID NO:1, amino acid residues 1-40 of SEQ ID NO:1, amino acidresidues 1-42 of SEQ ID NO:1, amino acid residues 10-30 of SEQ ID NO:1,amino acid residues 20-40 of SEQ ID NO:1, and amino acid residues 20-42of SEQ ID NO:1.

In another embodiment of the present invention, the Aβ peptide of thepolymerized product contains two amino acid substitutions at amino acidresidues 18 and 19 of SEQ ID NO:1. In one embodiment the amino acidsubstitutions comprise a valine to lysine substitution at position 18and a phenylalanine to lysine substitution at position 19 of SEQ IDNO:1. Peptides having these two amino acid substitutions are referred toas AβK₁₈K₁₉ peptides and are derived from the amino acid sequence of SEQID NO:2 below.

(SEQ ID NO: 2) daefrhdsgy evhhqklkkf aedvgsnkga iiglmvggvv ia

Accordingly, suitable Aβ peptides comprising the double lysinesubstitution include, without limitation, peptides comprising amino acidresidues 1-30 of SEQ ID NO:2 (Aβ1-30K₁₈K₁₉), amino acid residues 1-40 ofSEQ ID NO:2 (Aβ1-40K₁₈K₁₉), amino acid residues 1-42 of SEQ ID NO:2(Aβ1-42K₁₈K₁₉), amino acid residues 1-20 of SEQ ID NO:2 (Aβ1-20K₁₈K₁₉),amino acid residues 10-30 of SEQ ID NO:2 (Aβ10-30K₁₈K₁₉), amino acidresidues 10-40 of SEQ ID NO:2 (Aβ10-40K₁₈K₁₉), amino acid residues 10-42of SEQ ID NO:2 (Aβ10-42K₁₈K₁₉), amino acid residues 20-40 of SEQ ID NO:2(Aβ20-40K₁₈K₁₉), and amino acid residues 20-42 of SEQ ID NO:2(Aβ20-42K₁₈K₁₉).

The polymerized product of the present invention may comprise ahomopolymer of an Aβ peptide or a copolymer of two or more different Aβpeptides. Alternatively, the polymerized product of the presentinvention may comprise a copolymer of an Aβ protein or peptideco-polymerized with any one or more of an α-synuclein protein orpeptide, a tau protein or peptide, a TDP-43 protein or peptide, anamylin protein or peptide, and/or a PrP protein or peptide. In oneembodiment of the present invention, the polymerized product comprises acopolymer of one or more Aβ peptides copolymerized with one or moreα-synuclein proteins or peptides. In another embodiment of the presentinvention, the polymerized product comprises a copolymer of one or moreAβ peptides copolymerized with one or more tau proteins or peptides. Inanother embodiment of the present invention, the polymerized productcomprises a copolymer of one or more Aβ peptides copolymerized with oneor more TDP-43 proteins or peptides

The polymerized product of the present invention may also comprise anα-synuclein protein or peptide thereof. The α-synuclein proteincomprises the amino acid sequence of SEQ ID NO:3.

(SEQ ID NO: 3)   1mdvfmkglsk akegvvaaae ktkqgvaeaa gktkegvlyv gsktkegvvh gvatvaektk  61eqvtnvggav vtgvtavaqk tvegagsiaa atgfvkkdql gkneegapqe giledmpvdp 121dneayempse egyqdyepea

Accordingly, in one embodiment of the invention, the α-synuclein proteinof the polymerized product comprises amino acid residues 1-140 of SEQ IDNO:3. Peptides of α-synuclein derived from the amino acid sequence ofSEQ ID NO: 3 are also suitable for use in the polymerized product of thepresent invention. In one embodiment, the peptide comprises at leastfive contiguous amino acids of SEQ ID NO:3. In another embodiment, thepeptide comprises at least 10, at least 15, at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, at least 50, at least55, at least 60, at least 65, at least 70, at least 75, at least 80, atleast 90 or at least 100 contiguous amino acids of SEQ ID NO:3.

In one embodiment of the invention, the α-synuclein peptide is aC-terminal α-synuclein peptide as disclosed in WO 2004/041067 to Schenkat al., which is hereby incorporated by reference in its entirety.Suitable C-terminal α-synuclein peptides for use in the polymerizedproduct of the present invention include, without limitation, peptidescomprising amino acids residues 70-140 of SEQ ID NO:3, amino acidresidues 100-140 of SEQ ID NO:3, amino acid residues 120-140 of SEQ IDNO:3, amino acid residues 130-140 of SEQ ID NO:3, or amino acid residues135-140 of SEQ ID NO:3.

The polymerized product of the present invention may comprise ahomopolymer of an α-synuclein protein or peptide or a copolymer of twoor more different α-synuclein proteins or peptides. Alternatively, thepolymerized product of the present invention may comprise a copolymer ofan α-synuclein protein or peptide co-polymerized with any one or more ofan Aβ peptide, a tau protein or peptide, a TDP-43 protein or peptide, anamylin protein or peptide, and/or a PrP protein or peptide.

The pharmaceutical composition of the present invention may alsocomprise a tau protein or peptide thereof. Suitable tau proteins of thepresent invention include any of the eight isoforms of the human tauprotein that are known in the art (see NCBI Accession Nos. NP_058519.3,NP_005901, NP_058518.1, NP_058525.1, NP_001116539.1, NP_001116538.2,NP_001190180.1, and NP_001190181.1, which are hereby incorporated byreference in their entirety). The tau isoforms vary in the number ofN-terminal inserts resulting from the splicing of exons two and three,and microtubule-binding domains resulting from the splicing of exon ten.The amino acid sequence corresponding to human tau protein isoform 2(441a.a), containing two N-terminal inserts and four microtubule binding(2N4R) domains is provided as SEQ ID NO:4 below.

(SEQ ID NO: 4)Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1               5                   10                  15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His            20                  25                  30Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu        35                  40                  45Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser    50                  55                  60Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val65                  70                  75                  80Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu                85                  90                  95Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro            100                 105                 110Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val        115                 120                 125Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly    130                 135                 140Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro145                 150                 155                 160Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro                165                 170                 175Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly            180                 185                 190Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser        195                 200                 205Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys    210                 215                 220Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys225                 230                 235                 240Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val                245                 250                 255Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly            260                 265                 270Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln        275                 280                 285Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly    290                 295                 300Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser305                 310                 315                 320Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln                325                 330                 335Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser            340                 345                 350Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn        355                 360                 365Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala    370                 375                 380Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser385                 390                 395                 400Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser                405                 410                 415Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val            420                 425                 430Ser Ala Ser Leu Ala Lys Gln Gly Leu

Accordingly, in one embodiment, a tau protein of the polymerized productof the present invention comprises amino acid residues 1-441 of SEQ IDNO:4. Alternatively, the composition of the invention comprises aminoacid residues 1-758 of tau isoform 1 (NP_058519.3), amino acid residues1-383 of tau isoform 3 (NP_058518.1), amino acid residues 1-352 of tauisoform 4 (NP_058525.1), amino acid residues 1-412 of tau isoform 5(NP_001116539.1), amino acid residue 1-776 of tau isoform 6(NP_001116538.2), amino acid residues 1-381 of tau isoform 7(NP_001190180.1), and amino acid residues 1-410 of tau isoform 8(NP_001190181.1).

The pharmaceutical composition of the present invention may alsocomprise tau peptides derived from the amino acid sequence of SEQ IDNO:4 or the amino acid sequence of any other tau isoform. In oneembodiment, the tau peptide comprises at least five contiguous aminoacids of a tau protein, e.g., the tau protein of SEQ ID NO:4. In anotherembodiment, the tau peptide comprises at least 10, at least 15, at least20, at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 90, or at least 100 contiguous amino acids ofa tau protein, e.g., the tau protein of SEQ ID NO:4. In yet anotherembodiment, the peptide comprises at least 200, at least 300, at least400, or at least 500 contiguous amino acids, of a tau protein.

A number of tau peptides that are suitable for polymerization andincorporation into the polymerized product of the present invention aredisclosed in U.S. Pat. No. 8,012,936 to Sigurdsson et al., which ishereby incorporated by reference in its entirety. Suitable peptidesinclude, without limitation, amino acid residues 1-30 of SEQ ID NO:4,amino acid residues 30-60 of SEQ ID NO:4, amino acid residues 60-90 ofSEQ ID NO:4, amino acid residues 90-120 of SEQ ID NO:4, amino acidresidues 120-150 of SEQ ID NO:4, amino acid residues 150-180 of SEQ IDNO:4, amino acid residues 210-240 of SEQ ID NO:4, amino acid residues270-300 of SEQ ID NO:4, amino acid residues 300-330 of SEQ ID NO:4,amino acid residues 330-360 of SEQ ID NO:4, amino acid residues 360-390of SEQ ID NO:4, amino acid residues 390-420 of SEQ ID NO:4, amino acidresidues 411-440 of SEQ ID NO:4, amino acid residues 133-162 of SEQ IDNO:4, amino acid residues 379-409 of SEQ ID NO:4, amino acid residues192-221 of SEQ ID NO:4, amino acid residues 221-250 of SEQ ID NO:4, oramino acid residues 184-213 of SEQ ID NO:4.

The polymerized product of the present invention may comprise ahomopolymer of tau protein or peptide or a copolymer of two or moredifferent tau proteins or peptides. Alternatively, the polymerizedproduct of the present invention may comprise a copolymer of a tauprotein or peptide co-polymerized with any one or more of an Aβ peptide,an α-synuclein protein or peptide, a TDP-43 protein or peptide, anamylin protein or peptide, and/or a PrP protein or peptide.

The polymerized product of the present invention may also comprise a TARDNA-binding protein 43 (TDP 43) or peptide thereof. The human TDP 43protein comprises the amino acid sequence of SEQ ID NO:5.

(SEQ ID NO: 5)   1mseyirvted endepieips eddgtvllst vtaqfpgacg lryrnpvsqc mrgvrlvegi  61lhapdagwgn lvyvvnypkd nkrkmdetda ssavkvkrav qktsdlivlg lpwktteqdl 121keyfstfgev lmvqvkkdlk tghskgfgfv rfteyetqvk vmsqrhmidg rwcdcklpns 181kqsqdeplrs rkvfvgrcte dmtedelref fsqygdvmdv fipkpfrafa fvtfaddqia 241qslcgedlii kgisvhisna epkhnsnrql ersgrfggnp ggfgnqggfg nsrgggaglg 301nnqgsnmggg mnfgafsinp ammaaaqaal qsswgmmgml asqqnqsgps gnnqnqgnmq 361repnqafgsg nnsysgsnsg aaigwgsasn agsgsgfngg fgssmdskss gwgm

Accordingly, in one embodiment of the invention, the TDP-43 protein ofthe polymerized product comprises amino acid residues 1-414 of SEQ IDNO:5. Peptides of TDP-43 derived from the amino acid sequence of SEQ IDNO:5 are also suitable for use in the pharmaceutical composition of thepresent invention. In one embodiment, the TDP-43 peptide comprises atleast five contiguous amino acids of SEQ ID NO:5. In another embodiment,the TDP-43 peptide comprises at least 10, at least 15, at least 20, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 55, at least 60, at least 65, at least 70, at least 75, atleast 80, at least 90, at least 100, at least 150, at least 200, atleast 250, or at least 300 contiguous amino acids of SEQ ID NO:5.

The polymerized product of the present invention may comprise ahomopolymer of a TDP-43 protein or peptide or a copolymer of two or moredifferent TDP-43 proteins or peptides. Alternatively, the polymerizedproduct of the present invention may comprise a copolymer of a TDP-43protein or peptide co-polymerized with any one or more of an Aβ peptide,a tau protein or peptide, an α-synuclein protein or peptide, an amylinprotein or peptide, and/or a PrP protein or peptide.

The polymerized product of the present invention can also comprise anamylin protein or peptide thereof. The human amylin protein comprisesthe amino acid sequence of SEQ ID NO:6.

(SEQ ID NO: 6)  1hqvekrkcnt atcatqrlan flvhssnnfg ailsstnvgs ntygkrnave vlkreplnyl 61 pl

Accordingly, in one embodiment of the invention, the amylin protein ofthe polymerized product comprises amino acid residues 1-62 of SEQ IDNO:6. Peptides of amylin derived from the amino acid sequence of SEQ IDNO:6 are also suitable for use in the pharmaceutical composition of thepresent invention. In one embodiment, the amylin peptide comprises atleast five contiguous amino acids of SEQ ID NO:6. In another embodiment,the amylin peptide comprises at least 10, at least 15, at least 20, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 55, at least 60 contiguous amino acids of SEQ ID NO:6.

The polymerized product of the present invention may comprise ahomopolymer of an amylin protein or peptide or a copolymer of two ormore different amylin proteins or peptides. Alternatively, thepolymerized product of the present invention may comprise a copolymer ofan amylin protein or peptide co-polymerized with any one or more of anAβ peptide, a tau protein or peptide, an α-synuclein protein or peptide,a TDP-43 protein or peptide, and/or a PrP protein or peptide.

The polymerized product of the present invention may also comprise aprion protein or peptide thereof. The human prion protein comprises theamino acid sequence of SEQ ID NO:7.

(SEQ ID NO: 7)   1manlgcwmlv lfvatwsdlg lckkrpkpgg wntggsrypg qgspggnryp pqggggwgqp  61hgggwgqphg ggwgqphggg wgqphgggwg qgggthsqwn kpskpktnmk hmagaaaaga 121vvgglggyvl gsamsrpiih fgsdyedryy renmhrypnq vyyrpmdeys nqnnfvhdcv 181nitikqhtvt tttkgenfte tdvkmmervv eqmcitqyer esqayykrgs smvlfssppv 241illisflifl ivg

Accordingly, in one embodiment of the invention, the prion protein ofthe polymerized product comprises amino acid residues 1-253 of SEQ IDNO:7 or analogs thereof. Peptides of prion protein derived from theamino acid sequence of SEQ ID NO:7 are also suitable for use in thepolymerized product of the present invention. In one embodiment, thepeptide comprises at least five contiguous amino acids of SEQ ID NO:7.In another embodiment, the peptide comprises at least 10, at least 15,at least 20, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 55, at least 60, at least 65, at least70, at least 75, at least 80, at least 90 or at least 100, at least 125,and least 150, at least 200, or at least 225 contiguous amino acids ofSEQ ID NO:7.

The polymerized product of the present invention may comprise ahomopolymer of an PrP protein or peptide or a copolymer of two or moredifferent PrP proteins or peptides. Alternatively, the polymerizedproduct of the present invention may comprise a copolymer of an PrPprotein or peptide co-polymerized with any one or more of an Aβ peptide,a tau protein or peptide, an α-synuclein protein or peptide, a TDP-43protein or peptide, and/or an amylin protein or peptide as describedherein.

The polymerized product of the present invention may further compriseone or more polymerized ABri peptides (CSRTVKKNIIEEN; SEQ ID NO: 8),ADan peptides (CFNLFLNSQEKHY; SEQ ID NO:9), or ABri/ADan fusion peptides(CSRTVKKNIIEENGSGSGCFNLFLNSQEKHY; SEQ ID NO:10) as disclosed in U.S.Patent Application Publication No. 20100284909 to Wisniewski et al.,which is hereby incorporated by reference in its entirety.

The proteins and peptides of the polymerized product of the presentinvention may comprise naturally occurring peptides or analog peptides.Analog peptides typically differ from naturally occurring peptides atone, two, or a few positions, often by virtue of conservativesubstitutions. Analogs typically exhibit at least 80 or 90% sequenceidentity with natural peptides. Some analogs include unnatural aminoacids or modifications of N or C terminal amino acids at one, two or afew positions. Examples of unnatural amino acids are D-amino acids,alpha amino acids, alpha-disubstituted amino acids, N-alkyl amino acids,lactic acid, 4-hydroxyproline, gamma-carboxyglutamate,epsilon-N,N,N-trimethyllysine, epsilon-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,omega-N-methylarginine, β-alanine, ornithine, norleucine, norvaline,hydroxproline, thyroxine, gamma-amino butyric acid, homoserine,citrulline, and isoaspartic acid. Analog peptides can be screened forprophylactic or therapeutic efficacy using animal models as described inthe Examples herein.

In another embodiment of the present invention, the polymerized productcomprises a polymer of a fusion peptide. Suitable fusion peptidescontain any first Aβ, α-synuclein, tau, TDP-43, amylin, or prion proteinpeptide of the present invention fused to any second Aβ, α-synuclein,tau, TDP-43, amylin, or prion protein peptide of the present invention.The fusion peptide preferably contains a short linker sequence couplingthe first peptide to a second peptide. Preferred linker sequencesinclude glycine-rich (e.g. G₃₋₅) or serine-rich (e.g. GSG, GSGS (SEQ IDNO: 13), GSGSG (SEQ ID NO: 14), GS_(N)G) linker sequences.

In accordance with this aspect of the present invention the polymerizedproduct may contain a polymer or copolymer of any one or more of the Aβ,α-synuclein, tau, TDP-43, amylin, or prion protein proteins or peptidesdescribed herein further linked in-frame to an adjuvant polypeptide. Theadjuvant polypeptide can be any adjuvant polypeptide known in the art,including, but not limited to, cholera toxin B, flagellin, humanpapillomavirus L1 or L2 protein, herpes simplex glycoprotein D (gD),complement C4 binding protein, TL4 ligand, and IL-1β. The protein orpeptides comprising the polymers of the polymerized product of thepresent invention may be linked directly to the adjuvant polypeptide orcoupled to the adjuvant by way of a short linker sequence. Suitablelinker sequences include glycine or serine-rich linkers described supraor other flexible immunoglobulin linkers as disclosed in U.S. Pat. No.5,516,637 to Huang et al, which is hereby incorporated by reference inits entirety.

In another embodiment, the polymerized product of the present inventionmay contain a polymer or copolymer of any one of the Aβ, α-synuclein,tau, TDP-43, amylin, or prion protein proteins or peptides describedsupra conjugated to an immunogenic carrier molecule. The immunogeniccarrier molecule can be covalently or non-covalently bonded to theproteins or peptides as described herein. Suitable immunogenic carriermolecules include, but are not limited to, serum albumins, chicken eggovalbumin, keyhole limpet hemocyanin, tetanus toxoid, thyroglobulin,pneumococcal capsular polysaccharides, CRM 197, immunoglobulinmolecules, alum, and meningococcal outer membrane proteins. Othersuitable immunogenic carrier molecules include T-cell epitopes, such astetanus toxoid (e.g., the P2 and P30 epitopes), Hepatitis B surfaceantigen, pertussis, toxoid, diphtheria toxoid, measles virus F protein,Chlamydia trachomatis major outer membrane protein, Plasmodiumfalciparum circumsporozite T, P. falciparum CS antigen, Schistosomamansoni triose phosphate isomersae, Escherichia coli TraT, and Influenzavirus hemagluttinin (HA). Other suitable immunogenic carrier moleculesinclude promiscuous T helper cell epitopes which are derived fromhepatitis B virus, Bordetella pertussis, Clostridium tetani, Pertusariatrachythallina, E. coli, Chlamydia trachomatis, Diphtheria, P.falciparum, and Schistosoma mansoni (see U.S. Pat. No. 6,906,169 toWang; U.S. Patent Application Publication No. 20030068325 to Wang, andWO 2002/096350 to Wang, which are hereby incorporated by reference intheir entirety). Yet other suitable carriers include T-helper cellepitopes derived from tetanus toxin, cholera toxin B, pertussis toxin,diphtheria toxin, measles virus F protein, hepatitis B virus surfaceantigen, C. trachomatis major outer membrane protein, P. falciparumcircumsporozoite, S. mansoni triose phosphate isomerase, or E. coli TraT(see WO01/42306 to Chain, which is hereby incorporated by reference inits entirety).

The peptides and proteins of the present invention can be linked toimmunogenic carrier molecules by chemical crosslinking prior topolymerization. Techniques for linking a peptide immunogen to animmunogenic carrier molecule include the formation of disulfide linkagesusing N-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) andsuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (ifthe peptide lacks a sulfhydryl group, this can be provided by additionof a cysteine residue). These reagents create a disulfide linkagebetween themselves and peptide cysteine residues on one protein, and anamide linkage through the epsilon-amino on a lysine, or other free aminogroup in other amino acids. A variety of such disulfide/amide-formingagents are described by Jansen et al., “Immunotoxins: Hybrid MoleculesCombining High Specificity and Potent Cytotoxicity,” Immun Rev62:185-216 (1982), which is hereby incorporated by reference in itsentirety. Other bifunctional coupling agents form a thioether ratherthan a disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, 2-iodoacetic acid, and4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

The one or more proteins or peptides comprising the polymers orcopolymers of the polymerized product of the present invention can besynthesized by solid phase or solution phase peptide synthesis,recombinant expression, or can be obtained from natural sources.Automatic peptide synthesizers are commercially available from numeroussuppliers, such as Applied Biosystems, Foster City, Calif. Standardtechniques of chemical peptide synthesis are well known in the art (seee.g., SYNTHETIC PEPTIDES: A USERS GUIDE 93-210 (Gregory A. Grant ed.,1992), which is hereby incorporated by reference in its entirety).Protein or peptide production via recombinant expression can be carriedout using bacteria, such as E. coli, yeast, insect or mammalian cellsand expression systems. Procedures for recombinant protein/peptideexpression are well known in the art and are described by Sambrook etal, Molecular Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d ed.,1989).

Recombinantly expressed peptides can be purified using any one ofseveral methods readily known in the art, including ion exchangechromatography, hydrophobic interaction chromatography, affinitychromatography, gel filtration, and reverse phase chromatography. Thepeptide is preferably produced in purified form (preferably at leastabout 80% or 85% pure, more preferably at least about 90% or 95% pure)by conventional techniques. Depending on whether the recombinant hostcell is made to secrete the peptide into growth medium (see U.S. Pat.No. 6,596,509 to Bauer et al., which is hereby incorporated by referencein its entirety), the peptide can be isolated and purified bycentrifugation (to separate cellular components from supernatantcontaining the secreted peptide) followed by sequential ammonium sulfateprecipitation of the supernatant. The fraction containing the peptide issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the peptides from other proteins. Ifnecessary, the peptide fraction may be further purified by HPLC.

Polymerization of the proteins or peptides alone or conjugated to anadjuvant polypeptide or immunogenic carrier molecule can be achievedusing standard techniques known in the art. As used herein,polymerization refers to process of reacting two or more peptide and/orprotein units together under suitable conditions to formthree-dimensional networks or polymer chains. As described herein, theproteins and peptides can be polymerized by a reaction with a crosslinking reagent. Suitable cross-linking reagents include, but are notlimited to glutaraldehyde and1-Ethyl-3-[3-dimethylaminopropyllcarbodiimide hydrochloride (EDC) (seeGoni et al., “Immunomodulation Targeting Abnormal Protein ConformationReduced Pathology in a Mouse Model of Alzheimer's Disease,” PLoS One5(10):e13391 (2010), which is hereby incorporated by reference in itsentirety). Alternatively, the proteins and peptides can be polymerizedby cysteine oxidation induced disulfide cross linking.

Another aspect of the present invention is directed to a pharmaceuticalcomposition comprising the polymerized product of the invention and apharmaceutical carrier.

In accordance with this aspect of the present invention, thepharmaceutical composition may contain a single homopolymer of an Aβprotein or peptide, α-synuclein protein or peptide, tau protein orpeptide, TDP-43 protein or peptide, amylin protein or peptide, or prionprotein or peptide. Alternatively, the pharmaceutical composition maycontain a mixture of one or more proteins or peptides, i.e.heteropolymers of the one or more aforementioned proteins and/orpeptides.

The pharmaceutical composition of the present invention can furthercontain, in addition to peptide polymers, other pharmaceuticallyacceptable components (see REMINGTON'S PHARMACEUTICAL SCIENCE (19th ed.,1995), which is hereby incorporated by reference in its entirety). Theincorporation of such pharmaceutically acceptable components depends onthe intended mode of administration and therapeutic application of thepharmaceutical composition. Typically, however, the pharmaceuticalcomposition will include a pharmaceutically-acceptable, non-toxiccarrier or diluent, which are defined as vehicles commonly used toformulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the composition. Exemplary carriers or diluentsinclude distilled water, physiological phosphate-buffered saline,Ringer's solutions, dextrose solution, and Hank's solution.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized sepharose, agarose, cellulose), polymeric amino acids,amino acid copolymers, and lipid aggregates (such as oil droplets orliposomes).

The pharmaceutical composition of the present invention can furthercontain an adjuvant. One class of preferred adjuvants is aluminum salts,such as aluminum hydroxide, aluminum phosphate, or aluminum sulfate.Such adjuvants can be used with or without other specificimmunostimulating agents such as MPL or 3-DMP, QS-21, flagellin,attenuated Salmonella (e.g., Salmonella typhimurium), polymeric ormonomeric amino acids such as polyglutamic acid or polylysine, orpluronic polyols. Oil-in-water emulsion formulations are also suitableadjuvants that can be used with or without other specificimmunostimulating agents such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycerohydroxyphosphoryloxy)-ethylamine (MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™, or other bacterial cell wallcomponents). A suitable oil-in-water emulsion is MF59 (containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing variousamounts of MTP-PE) formulated into submicron particles using amicrofluidizer such as Model 110Y microfluidizer (Microfluidics, NewtonMass.) as described in WO90/14837 to Van Nest et al., which is herebyincorporated by reference in its entirety. Other suitable oil-in-wateremulsions include SAF (containing 10% Squalene, 0.4% Tween 80, 5%pluronic-blocked polymer L121, and thr-MDP, either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion) and Ribi™ adjuvant system (RAS; containing 2% squalene, 0.2%Tween 80, and one or more bacterial cell wall components). Another classof preferred adjuvants is saponin adjuvants, such as Stimulon™ (QS-21)or particles generated therefrom such as ISCOMs (immunostimulatingcomplexes) and ISCOMATRIX. Other suitable adjuvants include incompleteor complete Freund's Adjuvant (IFA), cytokines, such as interleukins(IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF),lysolecithin, tumor necrosis factor (TNF), and liposome polycation DNAparticles. Such adjuvants are generally available from commercialsources.

In another embodiment of the present invention, the pharmaceuticalcomposition further includes a delivery vehicle. Suitable deliveryvehicles include, but are not limited to biodegradable microspheres,microparticles, nanoparticles, liposomes, collagen minipellets, andcochleates.

In one embodiment of this aspect of the invention, the pharmaceuticalagent includes a mucosal delivery system. A preferred mucosal deliverysystem consists of attenuated Salmonella (e.g., Salmonella typhimurium)with a non-toxic fragment C of tetanus toxin (TetC) or glutathioneS-transferase (GST). Methods of mucosal vaccination via oraladministration of S. typhimurium are described in Goni et al., “MucosalVaccination Delays or Prevents Prion Infection via an Oral Route,”Neuroscience 133:413-21 (2005), and Goni et al., “High Titers of Mucosaland Systemic Anti-PrP Antibodies Abrogate Oral Prion Infection inMucosal-Vaccinated Mice,” Neuroscience 153:679-686 (2008), which arehereby incorporated by reference in their entirety.

Another aspect of the present invention relates to a method of inducingan immune response against an amyloidogenic form of a protein or peptidein a subject. This method involves administering to the subject apolymerized product of the present invention under conditions effectiveto induce an immune response against the amyloidogenic form of theprotein or peptide in the subject. In a preferred embodiment of thisaspect of the present invention, a subject that would benefit from animmune response against an amyloidogenic form of a protein or peptide isselected prior to administering the polymerized product.

As used herein, an “amyloid protein”, “amyloidogenic protein”, and“amyloidogenic form of a protein” are used interchangeably toencompasses any insoluble fibrous protein/peptide aggregate that isdeposited intra- or extracellularly within the body. Amyloidogenicprotein/peptide deposition may be organ-specific (e.g., central nervoussystem, pancreas, etc.) or systemic. In accordance with this aspect ofthe invention, amyloidogenic proteins/peptides subject to depositioninclude beta protein precursor, prion and prion proteins, α-synuclein,tau, ABri precursor protein, ADan precursor protein, amylin,apolipoprotein AI, apolipoprotein AII, lyzozyme, cystatin C, gelsolin,protein, atrial natriuretic factor, calcitonin, keratoepithelin,lactoferrin, immunoglobulin light chains, transthyretin, A amyloidosis,β2-microglobulin, immunoglobulin heavy chains, fibrinogen alpha chains,prolactin, keratin, and medin. Amyloid deposition may occur as its ownentity or as a result of another illness (e.g., multiple myeloma,chronic infection, or chronic inflammatory disease).

In accordance with this aspect of the present invention, an immuneresponse is the development of a beneficial humoral (antibody mediated)and/or a cellular (mediated by antigen-specific T cells or theirsecretion products) response directed against the polymerized,non-fibrillogenic proteins or peptides of the polymerized product andcross-reactive with any amyloidogenic protein. Such a response is anactive response induced by administration of the immunogenic polymerizedprotein and/or peptides and represents a therapeutic means for clearingor removing amyloid protein deposits from the body of the subject.

The presence of a humoral immunological response can be determined andmonitored by testing a biological sample (e.g., blood, plasma, serum,urine, saliva feces, CSF or lymph fluid) from the subject for thepresence of antibodies directed to the immunogenic component of theadministered polymerized product. Methods for detecting antibodies in abiological sample are well known in the art, e.g., ELISA, Dot blots,SDS-PAGE gels or ELISPOT. The presence of a cell-mediated immunologicalresponse can be determined by proliferation assays (CD4⁺ T cells) or CTL(cytotoxic T lymphocyte) assays which are readily known in the art.

The present invention is further directed to a method of preventingand/or treating a condition mediated by an amyloidogenic protein orpeptide in a subject. This method involves administering to the subject,a polymerized product of the present invention containing one or morepolymers or co-polymers comprising an amyloid-beta (Aβ) peptide, anα-synuclein protein or peptide, a tau protein or peptide, a TARDNA-binding protein 43 (TDP-43) protein or peptide, an amylin protein orpeptide, a prion protein (PrP) or peptide or any combination thereof asdescribed supra. The polymerized product is administered underconditions effective to treat the condition mediated by theamyloidogenic protein or peptide in the subject. In a preferredembodiment of this aspect of the present invention, a subject at risk ofhaving or having a condition mediated by an amyloidogenic protein orpeptide is selected prior to administering the polymerized product ofthe present invention.

Conditions or diseases associated with, or resulting from, thedeposition of amyloidogenic proteins or peptides include, but are notlimited to, Alzheimer's disease, diffuse Lewy body disease, Down'ssyndrome, fronto-temporal dementia, Parkinson's disease, hereditarycerebral hemorrhage with amyloidosis, kuru, Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker disease, fatal familial insomnia, Britishfamilial dementia, Danish familial dementia, familial cornealamyloidosis, Familial corneal dystrophies, medullary thyroid carcinoma,insulinoma, type 2 diabetes, isolated atrial amyloidosis, pituitaryamyloidosis, aortic amyloidosis, plasma cell disorders, familialamyloidosis, senile cardiac amyloidosis, inflammation-associatedamyloidosis, familial Mediterranean fever, dialysis-associatedamyloidosis, systemic amyloidosis, and familial systemic amyloidosis. Inaccordance with this aspect of the present invention, administration ofthe polymerized product is effective to stimulate an immune response inthe subject that is effective at reducing and/or clearing theamyloidogenic protein that is causing or exacerbating the aforementioneddisease conditions.

A second aspect of the present invention is directed to an isolatedantibody or binding portion thereof having antigenic specificity for anepitope a non-amyloidogenic, non-fibrillogenic polymerized product ofthe present invention. As described infra, the polymerized product ofthe present invention comprises two or more protein or peptide units,each unit independently selected from the group consisting of anamyloid-beta (Aβ) peptide, an α-synuclein protein or peptide, a tauprotein or peptide, a TAR DNA-binding protein 43 (TDP-43) protein orpeptide, an amylin protein or peptide, a prion protein (PrP) protein orpeptide, and any combination thereof.

As used herein, “epitope” refers to an antigenic determinant of the oneor more polymerized proteins or peptides of the present invention thatis recognized by the isolated antibody. The epitope recognized by theantibody of the present invention may be a linear epitope, i.e. theprimary structure of the amino acid sequence of the target proteins orpeptides. Preferably, the linear epitope recognized by the isolatedantibody of the present invention does not have amino acid sequencehomology to a non-amyloid protein. Alternatively, the epitope recognizedby the isolated antibody of the present invention is a non-linear orconformational epitope, i.e. the tertiary or quaternary structure of apolymerized protein or peptide. In one embodiment of the presentinvention, the non-linear or conformational epitope recognized by theisolated antibody of the present invention is a conformational epitopethat is common to or shared with one or more, or all, amyloidogenicproteins. Accordingly, the isolated antibody of the present inventionhas antigenic specificity for a shared conformational epitope common toall amyloidogenic proteins known in the art.

An isolated antibody of the present invention encompasses anyimmunoglobulin molecule that specifically binds to an epitope of apolymerized product of the present invention. Preferably, the antibodyof the present invention binds specifically to an epitope that is sharedby a polymerized product of the present invention and one or moreamyloidogenic proteins. As used herein, the term “antibody” is meant toinclude intact immunoglobulins derived from natural sources or fromrecombinant sources, as well as immunoreactive portions (i.e., antigenbinding portions) of intact immunoglobulins. The antibodies of thepresent invention may exist in a variety of forms including, forexample, polyclonal antibodies, monoclonal antibodies, intracellularantibodies (“intrabodies”), antibody fragments (e.g. Fv, Fab andF(ab)2), as well as single chain antibodies (scFv), chimeric antibodies,and humanized antibodies (Ed Harlow and David Lane, USING ANTIBODIES: ALABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 1999); Houstonet al., “Protein Engineering of Antibody Binding Sites: Recovery ofSpecific Activity in an Anti-Digoxin Single-Chain Fv Analogue Producedin Escherichia coli,” Proc Natl Acad Sci USA 85:5879-5883 (1988); Birdet al, “Single-Chain Antigen-Binding Proteins,” Science 242:423-426(1988)).

Antibodies of the present invention may also be synthetic antibodies. Asynthetic antibody is an antibody which is generated using recombinantDNA technology, such as, for example, an antibody expressed by abacteriophage. Alternatively, the synthetic antibody is generated by thesynthesis of a DNA molecule encoding and expressing the antibody of theinvention or the synthesis of an amino acid specifying the antibody,where the DNA or amino acid sequence has been obtained using syntheticDNA or amino acid sequence technology which is available and well knownin the art.

Methods for monoclonal antibody production may be carried out using thetechniques described herein or other well-known in the art (MONOCLONALANTIBODIES— PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A.Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporatedby reference in its entirety). Generally, the process involves obtainingimmune cells (lymphocytes) from the spleen of a mammal which has beenpreviously immunized with the antigen of interest (i.e., a polymerizedprotein or peptide product of the present invention) either in vivo orin vitro. Exemplary polymerized products comprising one or more Aβpeptides, α-synuclein proteins or peptides, tau proteins or peptides,TDP-43 proteins or peptides, amylin proteins or peptides, and a prionproteins or peptides are described supra.

The antibody-secreting lymphocytes are fused with myeloma cells ortransformed cells, which are capable of replicating indefinitely in cellculture, thereby producing an immortal, immunoglobulin-secreting cellline. Fusion with mammalian myeloma cells or other fusion partnerscapable of replicating indefinitely in cell culture is achieved bystandard and well-known techniques, for example, by using polyethyleneglycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation ofSpecific Antibody-Producing Tissue Culture and Tumor Lines by CellFusion,” Eur J Immunol 6:511 (1976), which is hereby incorporated byreference in its entirety). The immortal cell line, which may be murine,but may also be derived from cells of other mammalian species, isselected to be deficient in enzymes necessary for the utilization ofcertain nutrients, to be capable of rapid growth, and have good fusioncapability. The resulting fused cells, or hybridomas, are cultured, andthe resulting colonies screened for the production of the desiredmonoclonal antibodies. Colonies producing such antibodies are cloned,and grown either in vivo or in vitro to produce large quantities ofantibody.

Alternatively monoclonal antibodies can be made using recombinant DNAmethods as described in U.S. Pat. No. 4,816,567 to Cabilly et al, whichis hereby incorporated by reference in its entirety. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, for example, by RT-PCR using oligonucleotide primersthat specifically amplify the genes encoding the heavy and light chainsof the antibody. The isolated polynucleotides encoding the heavy andlight chains are then cloned into suitable expression vectors, whichwhen transfected into host cells such as E. coli cells, simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, and monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries (McCafferty et al., “Phage Antibodies: FilamentousPhage Displaying Antibody Variable Domains,” Nature 348:552-554 (1990);Clackson et al., “Making Antibody Fragments using Phage DisplayLibraries,” Nature 352:624-628 (1991); and Marks et al., “By-PassingImmunization. Human Antibodies from V-Gene Libraries Displayed onPhage,” J. Mol. Biol. 222:581-597 (1991), which are hereby incorporatedby reference in their entirety).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified using recombinant DNA technology to generate alternativeantibodies. For example, the constant domains of the light and heavychains of a mouse monoclonal antibody can be substituted for thoseregions of a human antibody to generate a chimeric antibody.Alternatively, the constant domains of the light and heavy chains of amouse monoclonal antibody can be substituted for a non-immunoglobulinpolypeptide to generate a fusion antibody. In other embodiments, theconstant regions are truncated or removed to generate the desiredantibody fragment of a monoclonal antibody. Furthermore, site-directedor high-density mutagenesis of the variable region can be used tooptimize specificity and affinity of a monoclonal antibody.

The monoclonal antibody of the present invention can be a humanizedantibody. Humanized antibodies are antibodies that contain minimalsequences from non-human (e.g., murine) antibodies within the variableregions. Such antibodies are used therapeutically to reduce antigenicityand human anti-mouse antibody responses when administered to a humansubject. In practice, humanized antibodies are typically humanantibodies with minimal to no non-human sequences.

An antibody can be humanized by substituting the complementaritydetermining region (CDR) of a human antibody with that of a non-humanantibody (e.g., mouse, rat, rabbit, hamster, etc.) having the desiredspecificity, affinity, and capability (Jones et al., “Replacing theComplementarity-Determining Regions in a Human Antibody With Those Froma Mouse,” Nature 321:522-525 (1986); Riechmann et al., “Reshaping HumanAntibodies for Therapy,” Nature 332:323-327 (1988); Verhoeyen et al.,“Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science239:1534-1536 (1988), which are hereby incorporated by reference intheir entirety). The humanized antibody can be further modified by thesubstitution of additional residues either in the Fv framework regionand/or within the replaced non-human residues to refine and optimizeantibody specificity, affinity, and/or capability.

The monoclonal Aβ of the present invention can also be a humanmonoclonal Aβ. A human antibody is an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human. Human antibodies can be produced using varioustechniques known in the art. Immortalized human B lymphocytes immunizedin vitro or isolated from an immunized individual that produce anantibody directed against a target antigen can be generated (See e.g.,Reisfeld et al., MONOCLONAL ANTIBODIES AND CANCER THERAPy 77 (Alan R.Liss ed., 1985) and U.S. Pat. No. 5,750,373 to Garrard, which are herebyincorporated by reference in their entirety). Also, the human antibodycan be selected from a phage library, where that phage library expresseshuman antibodies (Vaughan et al., “Human Antibodies with Sub-NanomolarAffinities Isolated from a Large Non-immunized Phage Display Library,”Nature Biotechnology, 14:309-314 (1996); Sheets et al., “EfficientConstruction of a Large Nonimmune Phage Antibody Library: The Productionof High-Affinity Human Single-Chain Antibodies to Protein Antigens,”Proc. Natl. Acad. Sci. U.S.A. 95:6157-6162 (1998); Hoogenboom et al.,“By-passing Immunisation. Human Antibodies From Synthetic Repertoires ofGermline VH Gene Segments Rearranged In Vitro,” J Mol Biol 227:381-8(1992); Marks et al., “By-passing Immunization. Human Antibodies fromV-gene Libraries Displayed on Phage,” J Mol Biol 222:581-97 (1991),which are hereby incorporated by reference in their entirety). Humanantibodies can also be made in transgenic mice containing humanimmunoglobulin loci that are capable upon immunization of producing thefull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. This approach is described in U.S. Pat. No.5,545,807 to Surani et al.; U.S. Pat. No. 5,545,806 to Lonberg et al.;U.S. Pat. No. 5,569,825 to Lonberg et al.; U.S. Pat. No. 5,625,126 toLonberg et al.; U.S. Pat. No. 5,633,425 to Lonberg et al.; and U.S. Pat.No. 5,661,016 to Lonberg et al., which are hereby incorporated byreference in their entirety

Procedures for raising polyclonal antibodies are also well known in theart. Typically, such antibodies are raised by administering thepolymerized product of the present invention subcutaneously to rabbits(e.g., New Zealand white rabbits), goats, sheep, swine, or donkeys whichhave been bled to obtain pre-immune serum. The polymerized product canbe injected in combination with an adjuvant. The rabbits are bledapproximately every two weeks after the first injection and periodicallyboosted with the same antigen three times every six weeks. Polyclonalantibodies are recovered from the serum by affinity chromatography usingthe corresponding polymerized product to capture the antibody. This andother procedures for raising polyclonal antibodies are disclosed in EdHarlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (ColdSpring Harbor Laboratory Press, 1988), which is hereby incorporated byreference in its entirety.

As noted above, in addition to whole antibodies, the present inventionencompasses binding portions of such antibodies. Such binding portionsinclude the monovalent Fab fragments, Fv fragments (e.g., single-chainantibody, scFv), and single variable V_(H) and V_(L) domains, and thebivalent F(ab′)₂ fragments, Bis-scFv, diabodies, triabodies, minibodies,etc. These antibody fragments can be made by conventional procedures,such as proteolytic fragmentation procedures, as described in JamesGoding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (AcademicPress, 1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORYMANUAL (Cold Spring Harbor Laboratory, 1988), which are herebyincorporated by reference in their entirety, or other methods known inthe art.

Also suitable for use in the present invention are antibody fragmentsengineered to bind to intracellular proteins, i.e. intrabodies. Althoughamyloid protein deposits are generally extracellular, intracellularaccumulation of certain amyloid proteins (e.g., Aβ1-42) have beenobserved (D'Andrea et al., “Targeting Amyloid Beta: TargetingIntracellular Aβ42 for Alzheimer's Disease Drug Discover,” DrugDevelopment Research 56:194-200 (2002); Knobloch et al., “IntracellularAbeta and Cognitive Deficits Precede beta-Amyloid Deposition in arcAbetaMice,” Neurobiol Aging 28(9):1297-306 (2007), which are herebyincorporated by reference in their entirety). Accordingly, an intrabodycan be used to bind selectively to an epitope of an amyloid proteinwithin a cell. In a preferred embodiment, the intrabody recognizes anepitope of the Aβ1-42 oligomer accumulating within the perikaryon ofaffected neurons (e.g., pyramidal neurons) in AD.

Intrabodies are generally obtained by selecting a single variable domainfrom variable regions of an antibody having two variable domains (i.e.,a heterodimer of a heavy chain variable domain and a light chainvariable domain). Single chain Fv fragments, Fab fragments, ScFv-Ckfusion proteins, single chain diabodies, V_(H)-C_(H)1 fragments, andeven whole IgG molecules are suitable formats for intrabody development(Kontermann R. E., “Intrabodies as Therapeutic Agents,” Methods34:163-70 (2004), which is here by incorporated by reference in itsentirety).

Intrabodies having antigen specificity for a conformational epitope ofan amyloidogenic protein can be obtained from phage display, yeastsurface display, or ribosome surface display. Methods for producinglibraries of intrabodies and isolating intrabodies of interest arefurther described in U.S. Published Patent Application No. 20030104402to Zauderer and U.S. Published Patent Application No. 20050276800 toRabbitts, which are hereby incorporated by reference in their entirety.Methods for improving the stability and affinity binding characteristicsof intrabodies are described in WO2008070363 to Zhenping;Contreras-Martinez et al., “Intracellular Ribosome Display via SecMTranslation Arrest as a Selection for Antibodies with Enhanced CytosolicStability,” J Mol Biol 372(2):513-24 (2007), which are herebyincorporated by reference in their entirety.

It may further be desirable, especially in the case of antibodyfragments, to modify the antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Antibody mimics are also suitable for use in accordance with the presentinvention. A number of antibody mimics are known in the art including,without limitation, those known as monobodies, which are derived fromthe tenth human fibronectin type III domain (¹⁰Fn3) (Koide et al., “TheFibronectin Type III Domain as a Scaffold for Novel Binding Proteins,” JMol Biol 284:1141-1151 (1998); Koide et al., “Probing ProteinConformational Changes in Living Cells by Using Designer BindingProteins: Application to the Estrogen Receptor,” Proc Natl Acad Sci USA99:1253-1258 (2002), each of which is hereby incorporated by referencein its entirety); and those known as affibodies, which are derived fromthe stable alpha-helical bacterial receptor domain Z of staphylococcalprotein A (Nord et al., “Binding Proteins Selected from CombinatorialLibraries of an alpha-helical Bacterial Receptor Domain,” NatureBiotechnol 15(8):772-777 (1997), which is hereby incorporated byreference in its entirety).

The present invention is further directed to a pharmaceuticalcomposition containing the isolated antibody of the present invention asdescribed supra. In a preferred embodiment, the isolated antibodyrecognizes and binds to a shared conformational epitope common to one ormore amyloid proteins. The pharmaceutical composition of the presentinvention may contain an antibody mixture where all antibodies recognizethe same conformational epitope. Alternatively, the pharmaceuticalcomposition may contain an antibody mixture where one or more antibodiesrecognize one or more different conformational epitopes of amyloidproteins. The pharmaceutical composition of the present inventionfurther contains a pharmaceutically acceptable carrier or otherpharmaceutically acceptable components as described supra.

Another aspect of the present invention relates to a method of treatinga condition mediated by an amyloidogenic protein in a subject. Thismethod involves administering to the subject an antibody of the presentinvention, where the antibody has antigen specificity for an epitope ofa non-amyloidogenic, non-fibrillogenic polymerized product, wherein thepolymerized product comprises two or more protein and/or peptide units,each unit independently selected from the group consisting of anamyloid-beta (Aβ) peptide, an α-synuclein protein or peptide, a tauprotein or peptide, a TAR DNA-binding protein 43 (TDP-43) protein orpeptide, an amylin protein or peptide, a prion protein (PrP) protein orpeptide, and any combination thereof. Preferably the antibody hasantigen specificity for a shared conformational epitope that is commonto one or more amyloidogenic proteins. The antibody or a pharmaceuticalcomposition containing the antibody is administered in an amounteffective to treat the condition involving the amyloidogenic protein inthe subject. In accordance with this aspect of the invention, theantibody or pharmaceutical composition containing the antibody isadministered in an amount effective to generate passive immunity in thesubject against one or more amyloidogenic proteins, thereby facilitatingthe clearance of amyloid deposits from the subject.

Conditions mediated by an amyloidogenic protein that are amenable totreatment in accordance with this aspect of the present invention aredescribed supra.

In a preferred embodiment of this aspect of the present invention, asubject having a condition or at risk of developing a condition mediatedby an amyloidogenic protein is selected prior to administration of theantibody of the present invention. Subjects amenable to treatment inaccordance with the methods of the present invention include individualsat risk of developing an amyloid related disease but not showingsymptoms, as well as subjects showing symptoms at the time oftherapeutic intervention (i.e. antibody administration). Diseasessubject to treatment include any disease associated with or caused by anamyloidogenic protein as described supra. The pharmaceuticalcompositions of the present invention contain polymerized products thatare not endogenous to the body, or antibodies specific for onlypathological protein conformations. Therefore, the risk of inducing anauto-immune response is avoided and prophylactic treatment using thesepharmaceutical compositions of the present invention is particularlysuitable.

In the case of Alzheimer's disease, for example, virtually anyone is atrisk of suffering from Alzheimer's disease if he or she lives longenough. Therefore, the compositions of the present invention can beadministered prophylactically to the general population without the needfor any assessment of the risk of the subject patient. However, thepresent methods and compositions are especially suitable forprophylactic treatment of individuals who have a known genetic risk ofAlzheimer's disease or other condition related to an amyloidogenicprotein. Genetic markers associated with a risk of Alzheimer's diseaseinclude mutations in the APP gene, particularly mutations at position717 and positions 670 and 671 referred to as the Hardy and Swedishmutations respectively. Other markers of risk are mutations in thepresenilin genes, PS1 and PS2, and mutations in ApoE4, family history ofAD, hypercholesterolemia, or atherosclerosis.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70 years of age. Treatment typicallyentails multiple dosages over a period of time. Treatment can bemonitored by assaying antibody, or activated T-cell or B-cell responsesto the therapeutic agent (e.g., polymerized product) over time. If theresponse falls, a booster dosage is indicated. In the case of potentialDown's syndrome patients, treatment can begin antenatally byadministering the therapeutic agent to the mother or shortly afterbirth.

In prophylactic applications, the pharmaceutical compositions of thepresent invention are administered to a patient susceptible to, orotherwise at risk of, a particular disease in an amount sufficient toeliminate or reduce the risk or delay the onset of the disease. Intherapeutic applications, pharmaceutical compositions are administeredto a patient suspected of, or already suffering from an amyloidogenicdisease in an amount sufficient to cure, or at least partially arrest,the symptoms of the disease and its complications. An amount adequate toaccomplish this is defined as a therapeutically- orpharmaceutically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. Typically, the immuneresponse is monitored and repeated dosages are given if the immuneresponse starts to fade.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the subject is a human, but insome diseases, such as prion protein related diseases, the subject canbe a nonhuman mammal, such as a bovine. Other non-human mammals amenableto treatment in accordance with the methods of the present inventioninclude primates, dogs, cats, rodents (e.g., mouse, rat, guinea pig),horses, deer, cervids, cattle and cows, sheep, and pigs. Treatmentdosages need to be titrated to optimize safety and efficacy, and couldinvolve oral treatment.

When treatment of a subject involves the administration of a polymerizedproduct of the present invention containing one or more polymerized orcopolymerized Aβ, α-synuclein, tau, TDP-43, amylin, and/or PrP proteinor peptide immunogens, the appropriate dosage will depend on whetheradjuvant is co-administered, with higher dosages being required in theabsence of adjuvant. The amount of an immunogen for administrationsometimes varies from 1 μg-500 μg per patient and more usually from5-500 μg per injection for human administration. Occasionally, a higherdose of 1-2 mg per injection is used. Typically about 10, 20, 50 or 100μg is used for each human injection. The timing of injections can varysignificantly from once a day, to once a week, to once a month, to oncea year, to once a decade. Generally an effective dosage can be monitoredby obtaining a fluid sample from the patient, generally a blood serumsample, and determining the titer of antibody developed against theimmunogen, using methods well known in the art and readily adaptable tothe specific antigen to be measured. Ideally, a sample is taken prior toinitial dosing and subsequent samples are taken and titered after eachimmunization. Generally, a dose or dosing schedule which provides adetectable titer at least four times greater than control or“background” levels at a serum dilution of 1:100 is desirable, wherebackground is defined relative to a control serum or relative to a platebackground in ELISA assays.

On any given day that a dosage of immunogen is given, the dosage isgreater than 1 μg/patient and usually greater than 10 μg/patient ifadjuvant is also administered, and greater than 10 μg/patient andusually greater than 100 μg/patient in the absence of adjuvant. Atypical regimen consists of an immunization followed by boosterinjections at 6 weekly intervals. Another regimen consists of animmunization followed by booster injections 1, 2 and 12 months later.Another regimen entails an injection every two months for life.Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

For passive immunization with a composition comprising an antibody ofthe present invention, the dosage ranges from about 0.0001 to 100 mg/kg,and more usually 0.01 to 5 mg/kg of the host body weight. An exemplarytreatment regime entails administration once per every two weeks or oncea month or once every 3 to 6 months. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated. Antibody is usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of antibody to polymerized proteinsor peptide in the patient. Alternatively, antibody can be administeredas a sustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody in the patient. In general, human antibodiesshow the longest half life, followed by humanized antibodies, chimericantibodies, and nonhuman antibodies. The dosage and frequency ofadministration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

Compositions of the present invention, i.e., polymerized products andantibodies, can be administered by parenteral, topical, intravenous,oral, subcutaneous, intraperitoneal, intranasal or intramuscular meansfor prophylactic and/or therapeutic treatment. The most typical route ofadministration for compositions formulated to induce an immune responseis subcutaneous although others can be equally effective. The next mostcommon is intramuscular injection. This type of injection is mosttypically performed in the arm or leg muscles. Intravenous injections aswell as intraperitoneal injections, intra-arterial, intracranial, orintradermal injections are also effective in generating an immuneresponse. In some methods, agents such as antibodies are injecteddirectly into a particular tissue where deposits have accumulated, forexample intracranial injection. Intramuscular injection or intravenousinfusion are preferred for administration of antibody. In some methods,particular therapeutic antibodies are injected directly into thecranium. In some methods, antibodies are administered as a sustainedrelease composition or device, such as a Medipad™ device.

The pharmaceutical agents of the present invention may be formulated forparenteral administration. Solutions or suspensions of the agent can beprepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof in oils. Illustrativeoils are those of petroleum, animal, vegetable, or synthetic origin, forexample, peanut oil, soybean oil, or mineral oil. In general, water,saline, aqueous dextrose and related sugar solution, and glycols, suchas propylene glycol or polyethylene glycol, are preferred liquidcarriers, particularly for injectable solutions. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

Pharmaceutical formulations suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

When it is desirable to deliver the pharmaceutical agents of the presentinvention systemically, they may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

Intraperitoneal or intrathecal administration of the agents of thepresent invention can also be achieved using infusion pump devices suchas those described by Medtronic, Northridge, Calif. Such devices allowcontinuous infusion of desired compounds avoiding multiple injectionsand multiple manipulations.

In addition to the formulations described previously, the compositionsof the present invention may also be formulated as a depot preparation.Such long acting formulations may be formulated with suitable polymericor hydrophobic materials (for example as an emulsion in an acceptableoil) or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Another aspect of the present invention relates to a method ofdiagnosing an amyloid disease in a subject. This method involvesdetecting, in the subject, the presence of an amyloidogenic proteins orpeptides using a diagnostic reagent, where the diagnostic reagent is anantibody, or active binding fragment thereof, of the present invention.As described supra, the antibody has antigenic specificity for aconformational epitope of an amyloidogenic form of a protein. Preferablythe conformational epitope recognized by the antibody is shared with oneor more other amyloidogenic protein or peptides. The diagnosis of theamyloid disease is based on the detection of an amyloidogenic protein orpeptide in the subject.

Detecting the presence of amyloidogenic deposits in a subject using thediagnostic reagent can be achieved by obtaining a biological sample fromthe subject (e.g., blood, urine, cerebral spinal fluid), contacting thebiological sample with the diagnostic antibody reagent, and detectingbinding of the diagnostic antibody reagent to an amyloidogenic proteinin the sample from the subject. Assays for carrying out the detection ofan amyloid protein in a biological sample using a diagnostic antibodyare well known in the art and include, without limitation, ELISA,immunohistochemistry, western blot.

Alternatively, detecting the presence of amyloid deposits in a subjectusing diagnostic antibody reagent of the present invention can beachieved using in vivo imaging techniques. In vivo imaging involvesadministering to the subject the diagnostic antibody having antigenicspecificity for a conformational epitope of a polymerized productcontaining two or more protein and/or peptide units where each unit isindependently selected from the group consisting of Aβ, α-synuclein,tau, TDP-43, amylin, and/or PrP proteins or peptides, and detecting thebinding of the diagnostic agent to the amyloidogenic protein in vivo. Asdescribed supra, preferred antibodies bind to a conformational epitopeof an amyloidogenic form of a protein or peptide without binding to thenon-amyloidogenic proteins and without binding to the non-pathologicalforms of the amyloidogenic proteins.

Diagnostic antibodies or similar reagents can be administered byintravenous injection into the body of the patient, or directly into thebrain by intracranial injection or by drilling a hole through the skull.The dosage of antibody should be within the same ranges as for treatmentmethods. Typically, the antibody is labeled, although in some methods,the primary antibody with affinity for the conformational epitope of anamyloid protein is unlabelled and a secondary labeling agent is used tobind to the primary antibody. The choice of label depends on the meansof detection. For example, a fluorescent label is suitable for opticaldetection. Use of paramagnetic labels is suitable for tomographicdetection without surgical intervention. Radioactive labels can also bedetected using PET or SPECT.

Diagnosis is performed by comparing the number, size, and/or intensityof labeled amyloid protein deposits in a sample from the subject or inthe subject, to corresponding baseline values. An appropriate baselinevalue can be the average level of amyloid protein deposition in apopulation of undiseased individuals. Alternatively, an appropriatebaseline value may be the level of amyloid protein deposition in thesame subject determined at an earlier time.

The diagnostic methods described above can also be used to monitor asubject's response to therapy. In this embodiment, detection of amyloiddeposits in the subject is determined prior to the commencement oftreatment. The level of amyloid deposition in the subject at thistimepoint is used as a baseline value. At various times during thecourse of treatment the detection of amyloid deposits can be repeated,and the measured values thereafter compared with the baseline values. Adecrease in values relative to baseline signals a positive response totreatment.

The present invention is further directed to a kit for performing theabove described diagnostic and monitoring methods. Typically, such kitscontain a diagnostic antibody reagent, preferably the antibody of thepresent invention that has antigenic specificity for a polymerized Aβ,α-synuclein, tau, TDP-43, amylin, and/or PrP protein or peptide product.The kit can also include a detectable label. The diagnostic antibodyitself may contain the detectable label (e.g., fluorescent molecule,biotin, etc.) which is directly detectable or detectable via a secondaryreaction (e.g., reaction with strepavidin). Alternatively, a secondreagent containing the detectable label may be utilized, where thesecond reagent has binding specificity for the primary antibody. In adiagnostic kit suitable for measuring amyloid in a biological sample,the antibodies of the kit may be supplied prebound to a solid phase,such as to the wells of a microtiter dish.

Diagnostic kits of the present invention also include kits that areuseful for detecting antibody production in a subject followingadministration of a polymerized protein or peptide of the presentinvention. Typically, such kits include a reagent that contains theantigenic epitope of the antibodies generated by the subject in apolymerized product as described supra. The kit also includes adetectable label. In a preferred embodiment, the label is typically inthe form of labeled anti-idiotypic antibodies. The antigenic epitopereagents of the kit can be supplied prebound to a solid phase, such asto the wells of a microtiter dish.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but they are by no means intended to limit its scope.

Materials and Methods for Examples 1-4

Peptide Synthesis: The 42 amino acid Aβ peptide (SEQ ID NO:1), the ABripeptide (Cys-Ser-Arg-Thr-Val-Lys-Lys-Asn-Ile-Ile-Glu-Glu-Asn) (SEQ IDNO:8), and Aβ1-30K₁₈K₁₉ (daefrhdsgy evhhqklkkf aedvgsnkga) (SEQ IDNO:12) were synthesized on an ABI 430A peptide synthesizer (AMEBioscience, Chicago, Ill.) at the Keck peptide synthesis facility atYale University, CT, using a Vydac C18 preparative column, 2.5×30 cm(Vydac Separations, Hesperia, Calif.). Standard protocols for tBOC(tert-butyloxycarbonyl) chemistry were used. The peptides weresubsequently cleaved from the resins using hydrofluoric acid andpurified by high-pressure liquid chromatography (HPLC) on a Vydac C18preparative column using linear gradients from 0-70% of acetonitrile in0.1% trifluoroacetic acid. Mass spectroscopy of the lyophilizedend-product was used to verify the expected molecular weight.

Peptide Polymerization and Assessment of Conformation. In order to makethe peptides immunogenic and to potentially ensure a conformationspecific immune response, the peptides were first subjected tocontrolled polymerization using the following protocol. Peptides weredissolved at 3 mg/ml, in 100 mM borate buffer saline (BBS), pH 7.4.Fresh 1% glutaraldehyde in BBS was prepared and added to the peptide toa final 5 mM glutaraldehyde concentration and incubated in an Eppendorfblock at 800 rpm at 56° C. for 16 hrs. The solution was then quenchedwith 0.5 M glycine to make the solution 100 mM in glycine. After fiveminutes the solution was diluted 1:3 with BBS, dialyzed against 2 mM BBSovernight at 4° C., aliquoted, and lyophilized.

For electron microscopic studies of the polymerized Aβ peptide, theoriginal and polymerized peptides were incubated at 1 mg/ml in phosphatebuffered saline, pH 7.4. The sample (3 μl) was put onto a carbon coated400 mesh Cu/Rh grid (Ted Pella Inc., Redding, Calif.) and stained with1% uranyl acetate in distilled water (Polysciences, Inc, Warrington,Pa.). Stained grids were examined under a Philips CM-12 electronmicroscope (FEI; Eindhoven, The Netherlands) and photographed with a (1k×1 k) digital camera (Gatan, Inc., Pleasanton, Calif.).

Immunization of Mice. Animal studies were approved by the NYU School ofMedicine Institutional Animal Care and Use Committee and were consistentwith the recommendations of the American Veterinary Association. Twotransgenic (Tg) mouse models were used in these experiments. The firstmodel, the 3×Tg model, develops both plaque and tangle pathology (Oddoet al., “Triple-Transgenic Model of Alzheimer's Disease with Plaques andTangles: Intracellular Abeta and Synaptic Dysfunction,” Neuron 39:409-21(2003), which is hereby incorporated by reference in its entirety). Thesecond model, the TgSwDI model, develops extensive congophilicangiopathy (CAA) (Davis et al., “Early-Onset and Robust CerebralMicrovascular Accumulation of Amyloid Beta-Protein in Transgenic MiceExpressing Low Levels of a Vasculotropic Dutch/Iowa Mutant Form ofAmyloid Beta-Protein Precursor,” J. Biol. Chem. 279:20296-306 (2004),which is hereby incorporated by reference in its entirety). Starting atthe age of 3 months mice were immunized 4 times biweekly, subcutaneouslywith 50 μg/animal of polymerized peptide in sterile saline:Alum 9:1, andthereafter 4 times bimonthly with 25 μg/animal until the 12 month. Atthe age of 15-16 months mice were subject to locomotor and cognitivebehavioral testing (radial arm maze), followed by histological andbiochemical analysis. Animals were bled from the caudal vein prior toinoculation (T0), after the 6^(th) inoculation (T6) and at the time ofsacrifice (TF). The blood was collected in heparinized tubes and plasmaseparated and stored at −80° C.

Locomotor and Cognitive Behavioral Testing. Locomotor Activity: AHamilton-Kinder Smart-frame Photobeam System was used to make acomputerized recording of animal activity over a designated period oftime. Exploratory locomotor activity is recorded in a circular openfield activity chamber measuring (70×70 cm). A video camera mountedabove the chamber automatically recorded horizontal movements in theopen field in each dimension (i.e., x, y, and two z planes). Totaldistance was measured in centimeters (cm) traveled and is defined assequential movement interruptions of the animal measured relative to thebackground. The duration of the behavior was timed for 15 min. Resultswere reported based on distance traveled (cm), mean resting time, andmaximum velocity of the animal.

Radial Arm Maze: Prior to testing, the mice were adapted to the roomwith lights on for 15 min. Spatial learning was evaluated using aneight-arm radial maze with a water well at the end of each arm. ClearPlexiglas guillotine doors, operated by a remote pulley system,controlled access to the arms from a central area from which the animalsentered and exited the apparatus. After 3-4 days of adaptation,water-restricted mice (2 hours daily access to water) were given onetraining session per day for ten consecutive days. For each session, allarms were baited with saccharine flavored water, and animals werepermitted to enter all arms until the eight rewards had been consumed.The number of errors (entries to previously visited arms) and time tocomplete each session were recorded.

Antibody Levels. Antibody levels were determined in duplicate on 1:100dilutions of plasma using ELISA as described previously (Goni et al.,“Mucosal Vaccination Delays or Prevents Prion Infection Via an OralRoute,” Neurosci. 133:413-421 (2005); Asuni et al., “Aβ DerivativeVaccination in Alum Adjuvant Prevents Amyloid Deposition and Does NotCause Brain Microhemorrhages in Alzheimer's Model Mice,” Eur. J.Neurosci. 24:2530-2542 (2006), which are hereby incorporated byreference in their entirety), in which 5 μg/plate Aβ1-40, Aβ1-42,Aβ1-30KK, or pABri was coated onto Immulon 2HB 96 well microtiter wells(Thermo, Waltham, Mass.). The bound antibodies were detected by ahorseradish peroxidase labeled goat anti-mouse IgG (AmershamBiosciences, Piscataway, N.J.) or a peroxidase conjugated goatanti-mouse IgM (Sigma; A8786). Tetramethyl benzidine (TMB; Pierce,Rockford, Ill.) was the color developing substrate and the readings weredone at 450 nm.

Histology. Mice were anesthetized with sodium pentobarbital (150 mg/kg,i.p.), perfused transaortically with phosphate buffer, and the brainsprocessed as described previously (Asuni et al., “Aβ DerivativeVaccination in Alum Adjuvant Prevents Amyloid Deposition and Does NotCause Brain Microhemorrhages in Alzheimer's Model Mice,” Eur. J.Neurosci. 24:2530-2542 (2006); Sigurdsson et al., “An Attenuated ImmuneResponse is Sufficient to Enhance Cognition in an Alzheimer's DiseaseMouse Model Immunized With Amyloid-β Derivatives,” J. Neurosci.24:6277-6282 (2004), which are hereby incorporated by reference in theirentirety). Serial coronal sections (40 μm) were stained with a mixtureof 4G8/6E10, monoclonal antibodies that recognizes Aβ and stains bothpre-amyloid and Aβ plaques (Sadowski et al., “Blocking theApolipoproteine/Amyloid β Interaction Reduces the Parenchymal andVascular Amyloid-β Deposition and Prevents Memory Deficit in ADTransgenic Mice,” Proc. Natl. Acad. Sci. (USA) 103:18787-18792 (2006);Scholtzova et al., “Induction of Toll-Like Receptor 9 Signaling as aMethod for Ameliorating Alzheimer's Disease Related Pathology,” J.Neurosci. 29:1846-1854 (2009), which are hereby incorporated byreference in their entirety). Immunostaining was performed as describedpreviously (Sadowski et al., “Blocking the Apolipoproteine/Amyloid βInteraction Reduces the Parenchymal and Vascular Amyloid-β Depositionand Prevents Memory Deficit in AD Transgenic Mice,” Proc. Natl. Acad.Sci. (USA) 103:18787-18792 (2006); Scholtzova et al., “Induction ofToll-Like Receptor 9 Signaling as a Method for Ameliorating Alzheimer'sDisease Related Pathology,” J. Neurosci. 29:1846-1854 (2009), which arehereby incorporated by reference in their entirety). All procedures wereperformed by an individual blinded to the experimental conditions of thestudy. The Aβ burden is defined as the percentage of area in themeasurement field occupied by reaction product.

Example 1—Locomotor and Cognitive Testing

In order to verify that cognitive testing was not confounded bydifferences in sensorimotor abilities in the polymerized peptidevaccinated versus control Tg3x and SwDI transgenic mice, locomotortesting was conducted first. There was no significant differencesbetween SwDI Tg control mice and SwDI Tg mice vaccinated withpolymerized Aβ1-30₁₈K₁₉K, polymerized ABri peptide (ABri-Glut), orpolymerized Aβ1-42 peptide in distance traveled (FIG. 1A) or meanvelocity (Vmean) (FIG. 1C). Aβ1-42 vaccinated Tg mice exhibited slowermaximum velocity (Vmax) (FIG. 1B) (p=0.0234 post-hoc only; Aβ1-42 versuscontrol p<0.05) and longer resting time (FIG. 1D) (p=0.0311 post-hoconly; Aβ1-42 versus control p<0.05) compared to controls. No significantdifferences between the Aβ1-30₁₈K₁₉K and ABri treated Tg animals versuscontrols in these parameters were observed (FIGS. 1B and 1D). There wasno significant difference between Tg3x control mice and Tg3x micevaccinated with a synthetic polymerized Aβ1-30K₁₈K₁₉, polymerized ABripeptide (ABri-Glut), or the combination of Aβ1-30 K₁₈K₁₉ and ABripolymerized peptides in distance traveled (FIG. 2A), maximum velocity(Vmax) (FIG. 2B), mean velocity (Vmean) (FIG. 2C), or in resting time(FIG. 2D).

Radial arm maze cognitive testing showed there were significantdifferences between the untreated control Tg3x mice (Tg control) versusAβri treated Tg mice and wild-type controls (FIG. 3A). There was nodifference between the wild-type controls and the ABri vaccinated Tg3xmice. There was also a significant difference between the untreatedcontrol Tg3x mice (Tg control) versus animals vaccinated with thepolymerized Aβ1-30K₁₈K₁₉ peptide and animals vaccinated with thecombination of polymerized ABri and Aβ1-30K₁₈K₁₉ peptides (FIG. 3B).

Radial arm maze cognitive testing in the SwDI Tg mice showed there weresignificant differences between the untreated control SwDI Tg mice (TgControl) versus the polymerized Aβ1-42, Aβ1-30K₁₈K₁₉, and Aβri treatedTg mice and wild-type controls (FIG. 3C). There was no differencebetween the wild-type controls and the vaccinated SwDI Tg mice.

Example 2—Antibody Titers

In TgSwDI mice vaccinated with polymerized ABri, significant IgG and IgMtiters were noted against Aβ1-40, Aβ1-42, and polymerized ABri at T1 andTf (FIG. 4A). In TgSwDI mice vaccinated with polymerized Aβ1-30K₁₈K₁₉,significant IgG and IgM titers were noted against Aβ1-40, Aβ1-42, andAβ1-30K₁₈K₁₉ at T1 (FIG. 4B). Significant IgM titers against Aβ1-40,Aβ1-42, and Aβ1-30 K₁₈K₁₉, and significant IgG titers against Aβ1-42were observed at Tf (FIG. 4B). In TgSwDI mice vaccinated withpolymerized Aβ1-42, significant IgG and IgM titers were also notedagainst Aβ1-40, Aβ1-42, and Aβ1-30K₁₈K₁₉ at T1, and to a lesser extentat Tf (see FIG. 4C).

In Tg3x mice vaccinated with polymerized ABri, significant IgG and IgMtiters were noted against Aβ1-42 and polymerized ABri at T1 and Tf (FIG.5A). Significant IgM titers against Aβ1-40 were observed at T1, andsignificant IgG titers against Aβ1-40 were observed at Tf in theseanimals. In the polymerized Aβ1-30K₁₈K₁₉ vaccinated Tg3x mice,significant IgG and IgM titers were noted against Aβ1-42 andAβ1-30K₁₈K₁₉ at T1 and Tf, and significant IgM and IgG antibodiesagainst Aβ1-40 were observed at Tf (FIG. 5B). In Tg3x animals vaccinatedwith the combination of polymerized Aβ1-30K₁₈K₁₉ and ABri, significantIgG and IgM titers were also noted against Aβ1-42, Aβ1-30K₁₈K₁₉, andABri at T1 and Tf (see FIG. 5C). FIG. 5D shows that IgG and IgM antibodytiter against Aβ1-40 and Aβ1-42 does not differ significantly in vehicletreated transgenic 3×Tg animals.

Example 3—Amyloid Burden

There was a significant reduction in hippocampal amyloid burden in Tg3xanimals administered polymerized ABri (Tg-pABri), polymerizedAβ1-30₁₈K₁₉K (Tg-AB1-30KK), or the combination of polymerized peptides(Tg-combined) compared to transgenic control (Tg-control) animals asshown in FIG. 6A. There was also a significant reduction in PHF1 in thehippocampus (FIG. 6B) and cortex (FIG. 6C), respectively, of Tg3xanimals administered polymerized ABri or the combination of polymerizedABri and Aβ1-30₁₈K₁₉K. No significant difference in PHF1 burden in thehippocampus or cortex was found between Tg-control and Tg-Aβ1-30₁₈K₁₉Ktreated animals.

There was a significant reduction in the hippocampal amyloid burden inSwDI Tg animals vaccinated with polymerized ABri, Aβ1-30K₁₈K₁₉, orAβ1-42 compared to control Tg mice (see FIG. 7A).

Example 4—Aβ1-42 Peptide Polymerization and Assessment of Conformation

To examine the stability of polymerized peptide conformation over time,aged Aβ1-42 was analyzed by electron microscopy. As shown in the EMphotomicrograph of FIG. 7B the polymerized Aβ1-42 peptide ispredominately in the form of spherical particles of ˜200 nm at 3 weekspost polymerization. Similarly, the EM photomicrograph of FIG. 8A showsthat Aβ1-42 peptide aged for 3 months after controlled polymerizationwith glutaraldehyde maintains the spherical particle form that istypical of oligomerized peptides/proteins without any evidence of fibrilformation. In contrast, a non-polymerized Aβ1-42 peptide from the samesynthesis batch as the peptide in FIG. 8A shows complete fibrillizationat 3 months.

Discussion of Examples 1-4

In summary, controlled polymerized forms of Aβ1-42 and Aβ1-30K₁₈K₁₉ havebeen obtained. As demonstrated by EM analysis the Aβ1-42 polymerizedform does not produce fibrils when aged. The polymerized peptides arestable immunogens that produce an immune response that more specificallytargets the oligomeric pathogenic form of Aβ without targeting thenormal, physiological conformers of Aβ. Vaccination of TgSwDI and Tg3xanimals with polymerized ABri, Aβ1-42 or Aβ1-30K₁₈K₁₉ elicits a goodantibody response and produces cognitive benefits. Administration ofpolymerized Aβ1-42 reduces amyloid beta pathology, reduces Aβ oligomerlevels, and diminishes congophilic angiopathy, which is extensive in theSwDI animal model. As demonstrated herein, polymeric synthetic peptideshave been designed that mimic the secondary structure found in amyloidand oligomeric forms of Aβ and pathological forms of tau. The antibodyresponse elicited can target both amyloid and tau pathology resulting ina cognitive benefit, pathology burden reduction, and lack of apparentautoimmune toxicity. Collectively, this data demonstrates thesepolymeric synthetic peptides are excellent candidates for comprehensiveimmunomodulation in AD.

Materials and Methods for Example 5

Five White Tail deer were inoculated (vaccinated group) orally with anattenuated salmonella carrying deer PrP to stimulate the mucosal immunesystem. At the same time six White Tail deer were inoculated with thesame strain of attenuated salmonella but without any insert or foreignprotein to be delivered (Control group). The inoculations were repeated4 times over a period of a few months following the protocol describedby Goni et al., “High Titers of Mucosal and Systemic Anti-PrP AntibodiesAbrogate Oral Prion Infection in Mucosal-Vaccinated Mice,” Neurosci.153:679-686 (2008), which is hereby incorporated by reference in itsentirety.

Before the first inoculation (T0) and after the fourth inoculation (T5)samples of serum, saliva, and feces were taken and the specificantibodies against PrP were measured by ELISA. Subsequently vaccinatedanimals were orally boosted with a mixture of recombinant polymerizeddeer PrP protein and polymerized PrP fragments corresponding to aminoacid residues 121-166; 13-155; 155-200 and 169-230 of the mouse prionprotein of SEQ ID NO: 11 below.

  1 manlgywlla lfvtmwtdvg lckkrpkpgg wntggs rypg qgspggnryp pqggtwgqph 61 gggwgqphgg swgqphggsw gqphgggwgq gggthnqwnk pskpktnlkh vagaaaagav121 vgglggymlg samsrpmihf gndwedryyr enmyrypnqv yyrpvdqysn qnnfvhdcvn181 itikqhtvtt ttkgenftet dvkmmervve qmcvtqyqke sqayydgrrs sstvlfsspp241 villisflif livg

The PrP peptides and recombinant protein were subject to controlledpolymerization as described herein and Goni et al, “ImmunomodulationTargeting Abnormal Protein Conformation Reduces Pathology in a MouseModel of Alzheimer's Disease,” PLoS One 5(10):e13391 (2010), which ishereby incorporated by reference in its entirety. The polymerized PrPfragments and PrP protein were dissolved in sterile saline and deliveredwith 9:1 sodium hydroxide (Alum).

Ten days after delivery of the first booster, serum, saliva, and fecessamples were taken (T6). Two months later the same boost withpolymerized PrP and polymerized PrP fragments was repeated. Ten daysafter the second booster, serum, saliva, and feces were collected (T7).

The animals were subsequently boosted again and then orally challengedby delivering Chronic Wasting Disease (CWD) brain homogenate in bait.Samples were taken at different times and survival was assessed to thetime the animals showed clear signs of prion disease.

Example 5—Administration of Polymerized PrP Protein/Peptides ProtectsAgainst Chronic Wasting Disease in Deer

FIGS. 9 and 10 show the anti-PrP IgA antibody titers in feces andanti-PrP IgM antibody titers in plasma, respectively, in both controland vaccinated deer over the course of the treatment regimen describedabove. The control group did not develop any noticeable IgA titerwhereas the vaccinated group showed some increase in mucosa titer afterthe immune response was established with the salmonella oral delivery.The IgA titers were initially very low (T5) but were greatly enhancedafter the animals were boosted with the polymerized PrP and PrPfragments (T6 and T7) showing the importance of these antigenicpreparation on generating a sustainable immune response (FIG. 9 ). Withregard to IgM antibody titers, the control group did not show anynoticeable increase in antibody titer; whereas the vaccinated groupshowed some concomitant IgM titer in serum at the same time of themucosal response (T5). Anti-PrP antibody titers in the vaccinatedanimals greatly increased after the two boosts with the polymerized PrPand PrP fragments showing that this type of boost could invoke atherapeutic serum response.

An analysis of the antibody specificity in the vaccinated deer foundthat the antibodies produced are specific to prion protein. FIG. 10contains immunoblot data using purified antibodies from T7 in vaccinatedanimal 781 and control animal 786. Lane 1 of the blot containssalmonella lysate, to which antibodies in both animals were highlyreactive to. Lanes 4 and 5 contain deer PrP and polymerized deer PrP.The bands appearing in both lanes of the blot incubated with antibodiesfrom the vaccinated animal but not in the lanes of the blot incubatedwith antibodies from the control animal indicate the generation andpresence of deer PrP antibodies in the vaccinated, but not controlanimals. Lanes 2 and 3 contain sheep PrP and polymerized sheep PrP. Theabsence of bands in these lanes indicates the specificity of theantibody response observed in the vaccinated animals, i.e., thegenerated antibodies are specific for deer PrP.

PrP vaccination was clearly protective as indicated by the Kaplan Meiersurvival curve of FIG. 12 . Both vaccinated and control animals werechallenged with exposure to CWD. Three out of the six control animalsbecame with sick prionoses CWD and had to be properly euthanized. Incontrast, none of the vaccinated animals had any signs of disease overthe course of 18 months after challenge.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A non-amyloidogenic, non-fibrillogenic polymerized product comprisingtwo or more protein and/or peptide units, wherein each unit isindependently selected from the group consisting of an amyloid-beta (Aβ)peptide, an α-synuclein protein or peptide, a tau protein or peptide, aTAR DNA-binding protein 43 (TDP-43) protein or peptide, an amylinprotein or peptide, a prion protein (PrP) protein or peptide, and anycombination thereof.
 2. The polymerized product of claim 1, wherein theAβ peptide comprises an amino acid sequence selected from the groupconsisting of amino acid residues 1-16 of SEQ ID NO:1, amino acidresidues 1-20 of SEQ ID NO: 1, amino acid residues 1-30 of SEQ ID NO:1,amino acid residues 1-40 of SEQ ID NO:1, amino acid residues 1-42 of SEQID NO:1, amino acid residues 10-30 of SEQ ID NO:1, amino acid residues20-40 of SEQ ID NO:1, and amino acid residues 20-42 of SEQ ID NO:1. 3.The polymerized product of claim 2, wherein the Aβ peptide comprisesamino acid residues 1-42 of SEQ ID NO:1.
 4. The polymerized product ofclaim 1, wherein the Aβ peptide comprises an amino acid sequenceselected from the group consisting of amino acid residues 1-30 of SEQ IDNO:2 (Aβ1-30K₁₈K₁₉), amino acid residues 1-40 of SEQ ID NO:2(Aβ1-40K₁₈K₁₉), amino acid residues 1-42 of SEQ ID NO:2 (Aβ1-42K₁₈K₁₉),amino acid residues 1-20 of SEQ ID NO:2 (Aβ1-20K₁₈K₁₉), amino acidresidues 10-30 of SEQ ID NO:2 (Aβ10-30K₁₈K₁₉), amino acid residues 10-40of SEQ ID NO:2 (Aβ10-40K₁₈K₁₉), amino acid residues 10-42 of SEQ ID NO:2(Aβ10-42K₁₈K₁₉), amino acid residues 20-40 of SEQ ID NO:2(Aβ20-40K₁₈K₁₉), and amino acid residues 20-42 of SEQ ID NO:2(Aβ20-42K₁₈K₁₉).
 5. The polymerized product of claim 4, wherein the Aβpeptide comprises amino acid residues 1-30 of SEQ ID NO:2(Aβ1-30K₁₈K₁₉).
 6. The polymerized product of claim 1, wherein theα-synuclein protein or peptide comprises an amino acid sequence of SEQID NO:3 or a peptide derived from the amino acid sequence of SEQ IDNO:3.
 7. The polymerized product of claim 1, wherein the tau protein orpeptide comprises an amino acid sequence of SEQ ID NO: 4 or a peptidederived from the amino acid sequence of SEQ ID NO:4.
 8. The polymerizedproduct of claim 1, wherein the TDP-43 protein or peptide comprises anamino acid sequence of SEQ ID NO:5 or a peptide derived from the aminoacid sequence of SEQ ID NO:5.
 9. polymerized product of claim 1, whereinthe amylin protein or peptide comprises an amino acid sequence of SEQ IDNO:6 or a peptide derived from the amino acid sequence of SEQ ID NO:6.10. The polymerized product of claim 1, wherein the PrP protein orpeptide comprising an amino acid sequence of SEQ ID NO:7 or a peptidederived from the amino acid sequence of SEQ ID NO:7.
 11. The polymerizedproduct of claim 1, wherein the polymerized product is a co-polymercomprising one or more amyloid-beta (Aβ) peptides copolymerized with oneor more α-synuclein proteins or peptides.
 12. The polymerized product ofclaim 1, wherein the polymerized product is a co-polymer comprising oneor more amyloid-beta (Aβ) peptides co-polymerized with one or more taupeptides.
 13. The polymerized product of claim 1, wherein thepolymerized product is a co-polymer comprising one or more TDP-43peptides co-polymerized with one or more amyloid-beta (Aβ) peptidesand/or one or more tau peptides.
 14. The polymerized product of claim 1,wherein an adjuvant polypeptide is linked in frame to one or more of theproteins or peptides of the polymerized product.
 15. The polymerizedproduct of claim 14, wherein the adjuvant polypeptide is selected fromthe group consisting of cholera toxin B, flagellin, human papillomavirusL1 or L2 protein, herpes simplex glycoprotein D (gD), complement C4binding protein, TL4 ligand, and IL-1β.
 16. (canceled)
 17. Thepolymerized product of claim 1, wherein an immunogenic carrier moleculeis conjugated to the one or more proteins or peptides of the polymerizedproduct.
 18. The polymerized product of claim 17, wherein theimmunogenic carrier molecule is covalently or non-covalently bonded tothe one or more proteins or peptides of the polymerized product.
 19. Thepolymerized product of claim 17, wherein the immunogenic carriermolecule is selected from the group consisting of bovine serum albumin,chicken egg ovalbumin, keyhole limpet hemocyanin, tetanus toxoid,diphtheria toxoid, thyroglobulin, a pneumococcal capsularpolysaccharide, CRM 197, and a meningococcal outer membrane protein. 20.A pharmaceutical composition comprising: the polymerized product ofclaim 1 and a pharmaceutically acceptable carrier.
 21. Thepharmaceutical composition of claim 20 further comprising: an adjuvant.